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CN106119164A - A kind of method of synthetic medium separation and Culture avain tuberculosis mycobacteria - Google Patents

A kind of method of synthetic medium separation and Culture avain tuberculosis mycobacteria Download PDF

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CN106119164A
CN106119164A CN201610517944.5A CN201610517944A CN106119164A CN 106119164 A CN106119164 A CN 106119164A CN 201610517944 A CN201610517944 A CN 201610517944A CN 106119164 A CN106119164 A CN 106119164A
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culture
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tuberculosis mycobacteria
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CN106119164B (en
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朱良全
张阁
蒋卉
冯宇
丁家波
彭小薇
孙翠丽
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China Institute of Veterinary Drug Control
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Abstract

A kind of method that the present invention relates to synthetic medium separation and Culture avain tuberculosis mycobacteria.Avain tuberculosis mycobacteria is cultivated and generally uses Pei Shi (Petragnani) solid medium, the quality of this fresh skim milk of culture medium main component and egg is affected greatly by factors such as milch cow and egg source, freshness, holding times, causes quality poor stability between batch.Additionally itself makes loaded down with trivial details, and the laboratory for fresh milk source of drawing material difficulty is difficult to carry out.And synthetic medium provided by the present invention and preparation method thereof, overcoming above defect, the culture medium being prepared as substantially reduces avain tuberculosis mycobacteria incubation time than Pei Shi culture medium, and is clinically separated effect and is substantially better than Pei Shi culture medium.There is advantage easy and simple to handle, stay-in-grade, be suitable for being clinically separated cultivation avain tuberculosis mycobacteria, provide sound assurance for fowl tuberculosis diagnosis.

Description

A kind of method of synthetic medium separation and Culture avain tuberculosis mycobacteria
Present disclosure relates to a kind of method of synthetic medium separation and Culture avain tuberculosis mycobacteria, belongs to Veterinary microbiology field.
Background technology
Fowl tuberculosis is (also known as Mycobacterium avium, to be seen China Veterinery Drug Inspection Office, China beast by avain tuberculosis mycobacteria Doctor Microbiological Culture Collection administrative center writes, China's veterinary's strain catalogue second edition 2002, and Scientia Agricultura Sinica technology is published Society, 2002, p83) a kind of infectious disease of poultry and bird is caused.Feature is that the histoorgans such as the liver of disease fowl, spleen and intestinal form meat Bud swells and cheesy calcium scoring.Primary disease can infringer and many animals.OIE is classified as B class animal epidemic disease Disease, and China is set to three class animal epidemics.This disease feature is chronic process, gradual become thin, anemia, cockscomb atrophy, walk lamely with And lay eggs minimizing or stopping.The course of disease continues 2~3 months, sometimes up to 1 year.Sick fowl is because of exhaustion or unexpected because liver degeneration ruptures Dead.The most incoming poultry-farm, then long-term existence, be difficult to eradicate.Fowl tuberculosis in various degree is had to occur in recent years during the whole nation, Sickness rate is high, and harm is serious, and economic loss is huge, and urgent needs sets up a kind of easy isolated culture method, in order to clinical beast Doctor and researcher in time, accurately carry out diagnosis, thus carry out epidemic situation the most effectively disposing.
Being successfully separated out avain tuberculosis mycobacteria in suspected lesion tissue is diagnosis fowl tuberculosis " goldstandard ".Fowl is tied Core mycobacterium generally includes four subspecies, and the first subspecies are avain tuberculosis mycobacterium serotype 1,2,3 and genotype IS901+ And IS1245+;Second subspecies have avain tuberculosis mycobacterium people/pig subspecies (hominissuis) serotype 4,5,6,8,9,10,11 With 21 and genotype IS901-And IS1245+;3rd subspecies are avain tuberculosis mycobacterium paratuberculosis subspecies;4th subspecies are fowl knot Core mycobacterium silvaticum (translate, and OIE terrestrial animal is examined by Ministry of Agriculture veterinary office/China Animal Health and Epidemiology Center Disconnected test and vaccine handbook, 2010).
Currently, isolated in China avain tuberculosis mycobacteria mainly uses Pei Shi (Petragnani) solid medium (China beast Doctor's Culture Collection, China Veterinery Drug Inspection Office compile, China's veterinary microorganism strain catalogue, and Chinese agriculture is published Society, 2002, hereinafter referred to as " strain catalogue ";The Ministry of Agriculture of the People's Republic of China, MOA, People's Republic of China's veterinary biologics Code, 2000 editions, Chinese agriculture publishing house, calendar year 2001, hereinafter referred to as " code ").This culture medium is mainly composed of fresh defat Milk and egg, its quality is affected greatly by factors such as milch cow and egg source, freshness, holding times, causes culture medium to criticize Quality poor stability between secondary.During additionally Pei Shi culture medium makes, fresh milk need to be gathered and carry out defat, and Rhizoma Solani tuber osi need to be prepared Immersion also separates egg yolk, and complex manufacturing technology is loaded down with trivial details.Additionally, the laboratory for fresh milk source of drawing material difficulty cannot at all Realize culture medium to make.Thus seriously restrict and have impact on nationwide diagnosis and prevention and control in time to fowl tuberculosis.
Summary of the invention
The purpose of the present invention
Technical solution of the present invention
1., by a method for synthetic medium separation and Culture avain tuberculosis mycobacteria, it is characterized in that production fowl type tuberculosis The avain tuberculosis mycobacteria reference culture suitable normal saline dilution of C68201, C68202 and C68203 freeze-drying lactobacillus of rhzomorph After, inoculate with defatted milk powder, Ovum Gallus domesticus Flavus lecithin in the solid medium of synthetic medium primary raw material, cultivate 7 for 37 DEG C, make For preparation fowl type tuberculin first order seed.
2., according to the isolated culture method of a kind of avain tuberculosis mycobacteria described in claim 1, it is characterized in that wherein institute The formula (W/V) of use synthetic medium is: defatted milk powder 20g, PPLO agar powder 20g, yolk lecithin 10g, asparagine 2.6g, peptone 2g, yeast powder 2g, glycerol 36ml, vitamin K 0.1g, malachite green oxalate 0.6g, dehydrated alcohol 20ml, deionization Water 1000ml.Its compound method is: PPLO agar powder and malachite green oxalate 500ml are gone after water heating is fully dissolved, 116 DEG C Sterilizing 15min, is cooled to 60 DEG C as A liquid;Defatted milk powder, asparagine, peptone, yeast powder, glycerol, vitamin K are used 500ml deionized water, after 50 DEG C of heating in water bath dissolve, 0.22 μm filtration sterilization is as B liquid;By yolk lecithin with sterile working Mode fully dissolve with the dehydrated alcohol of 20ml in sterile vessel after as C liquid, then by A liquid and B liquid and C liquid, (V/V is After 500:500:20) being sufficiently mixed, make culture medium slant with 8ml/ pipe and use.
The detailed description of the present invention
1. avain tuberculosis rhzomorph production strain
(1) source C68201/C68202/C68203 strain avain tuberculosis mycobacteria, China Veterinery Drug Inspection Office identify, Keeping and supply.
(2) cultural character and virulence
By each 1 of C68201/C68202/C68203 strain avain tuberculosis mycobacteria freeze-drying lactobacillus, use 1ml normal saline respectively After dissolved dilution, respectively propping up inoculation with 0.2ml/ is main synthetic medium with defatted milk powder, PPLO agar powder and Ovum Gallus domesticus Flavus lecithin Raw-material culture medium slant, cultivates 7 for 37 DEG C, and media surface grows up to yellowish white butyrous, the allusion quotation of bacterium colony thickness Type bacterium colony, scrapes 10mg viable bacteria intramuscular injection or takes each 5 of viable bacteria 1mg intravenous injection 8 week old SPF chicken, clinical observation 4 from inclined-plane Week, equal 5/5 morbidity or dead, it is suitable for breeding fowl type tuberculin and produces seed use.
2 culture medium use the synthetic medium of present invention design
(1) formula (W/V) is as follows:
Defatted milk powder 20g, PPLO agar powder 20g, yolk lecithin 10g, asparagine 2.6g, peptone 2g, yeast powder 2g, glycerol 36ml, malachite green oxalate 0.6g, micro-element K 0.1g, dehydrated alcohol 20ml, deionized water to 1000ml.
(2) synthetic medium compound method
PPLO agar powder and malachite green oxalate 500ml are gone after water heating is fully dissolved, 116 DEG C of sterilizing 15min, cooling To 60 DEG C as A liquid;By defatted milk powder, asparagine, peptone, yeast powder, glycerol, vitamin K 500ml deionized water, After 50 DEG C of heating in water bath dissolve, 0.22 μm filtration sterilization is as B liquid;By yolk lecithin in the way of sterile working at aseptic device As C liquid after ware fully dissolves with the dehydrated alcohol of 20ml, then A liquid and B liquid and C liquid (V/V is 500:500:20) are filled After dividing mixing, make culture medium slant with 8ml/ pipe standby.
(3) synthetic medium inspection
1) character is faint yellow solid.
2) steriling test by People's Republic of China's veterinary drug allusion quotation (Chinese veterinary pharmacopoeia committee. People's Republic of China's veterinary drug Allusion quotation 2005 editions three. Chinese agriculture publishing house, 2006, hereinafter referred to as " Chinese veterinary pharmacopoeia ") method that specifies carries out, should be aseptic Growth.
3) growth test
By avain tuberculosis mycobacteria (C68201/C68202/C68203 strain, China Veterinery Drug Inspection Office provides) freeze-dried vaccine Planting respectively with after 1ml normal saline dilution, prop up inoculation synthetic medium solid slope with 0.2ml/, cultivate 7 for 37 DEG C, bacterium colony is Yellowish white butyrous, bacterium colony thickness, picking colony can be in wire drawing shape.As first order seed after purely passed examination.2~ 8 DEG C of preservations, should be less than (" code ") on the 45th.
The present invention relates to microbial resources information
The microbial resources related in the present invention are avain tuberculosis mycobacteria C68201 strain, C68202 strain and C68203 strain, Identified by China Veterinery Drug Inspection Office, take care of and supply and (ask for an interview China Veterinery Drug Inspection Office, China's veterinary microorganism strain Preservation administrative center writes, China's veterinary's strain catalogue second edition 2002, Scientia Agricultura Sinica technology publishing house, and 2002, p83).
The positive effect of the present invention
From doubtful fowl tuberculosis tissue, carry out bacteria distribution, be diagnosis fowl tuberculosis " goldstandard ".Isolated in China is trained Supporting avain tuberculosis mycobacteria and generally use Pei Shi (Petragnani) solid medium, this culture medium main component is fresh de- The quality of fat milk and egg is affected greatly by factors such as milch cow and egg source, freshness, holding times, causes batch interstitial Amount poor stability.Additionally itself makes loaded down with trivial details, and the laboratory for fresh milk source of drawing material difficulty is difficult to carry out, thus Affect avain tuberculosis mycobacteria separation and Culture and the diagnosis of clinical fowl tuberculosis.And synthetic medium provided by the present invention and Preparation method, overcomes above defect, and the culture medium being prepared as substantially reduces the training of avain tuberculosis mycobacteria than Pei Shi culture medium Support the time (can reduce by more than 3 days incubation times), and be clinically separated effect and be substantially better than Pei Shi culture medium.Efficiently solve new Fresh milk source of drawing material is difficult, technique makes a complicated difficult problem.There is advantage easy and simple to handle, stay-in-grade, be suitable for being clinically separated Cultivate avain tuberculosis mycobacteria, provide sound assurance for fowl tuberculosis diagnosis.
Embodiment
Embodiment 1
The research of avain tuberculosis mycobacteria synthetic medium
From 1%~3% defatted milk powder, 0.5%~1.5% yolk lecithin, 1%~2%PPLO agar powder be combined into In formula, by the avain tuberculosis mycobacteria (C68201/C68202/C68203 strain) of inoculated and cultured lyophilizing, filter out 2% defat The synthetic medium formula of milk powder+1% yolk lecithin+2%PPLO agar powder.This formula application result, 37 DEG C of quiescent culture 7 Day, avain tuberculosis mycobacteria (C68201/C68202/C68203 strain) media surface all grows up to yellowish white butyrous, bacterium colony The colonies typical of thickness.From cultivating inclined-plane scraping 10mg viable bacteria intramuscular injection or taking viable bacteria 1mg intravenous injection 8 week old SPF chicken each 5 Only, clinical observation 4 weeks, equal 5/5 morbidity or dead, basically identical with the bacterium result of the same race that Pei Shi solid medium is cultivated.Meet The requirement of " fowl type tuberculin manufacture strain " in " code ".
1 material
1.1 culture medium raw materials
(lot number 232100, purchased from BD Difco for skim milkTMCompany);(lot number 214010, is purchased from yolk lecithin OXOID company);(lot number: 214230, purchased from BD Difco for PPLO agar powderTMCompany);Pancreas casein peptone (lot number VM732731- 644, purchased from MERCK company);Yeast leaching powder (lot number 911948, purchased from OXOID company);Asparagine, glycerol, malachite green oxalate, Dehydrated alcohol, glucose, sucrose, sulfur are conventional chemical reagent for ` sodium sulfate, chlorhematin, vitamin K etc., purchased from north Capital chemical reagents corporation.
1.2 fresh milks (same day picks up from cattle farm, Beijing), egg, Rhizoma Solani tuber osi, potato starch surpass purchased from Beijing City, normal saline (lot number: 0619,4.5ml/ props up, 100ml/ bottle), A type test tube uses specification 25ml, China's veterinary medicament supervision Basis set offer is provided.
1.3 strain
The strain of production fowl type tuberculin: fowl type mycobacterium tuberculosis (C68201/C68202/C68203 strain) (2006.8.29 lyophilizing, 0.3ml/ props up), is identified by China Veterinery Drug Inspection Office, takes care of and supply.
2 methods and result
Prepared by 2.1 avain tuberculosis mycobacteria seed liquor
Carry out with reference to " code ", take each 1 of avain tuberculosis mycobacteria C68201, C68202 and C68203 freeze-drying lactobacillus, point Yong not prop up, with 0.2ml/, the Pei Shi culture medium that switching is prepared by 2.2 methods, cultivate 20 for 37 DEG C after 1ml normal saline dilution, As first order seed, wash lower surface lawn with appropriate normal saline the most respectively, be placed in containing in sterilizing glass bulb, use physiology Salt-water Oscillator mixing is diluted to Maxwell than turbid concentration about 106The bacterium solution of CFU/ml, uses as seed liquor.
2.2 Pei Shi (Petragnani) solid medium
2.2.1 composition: fresh skim milk 450ml, potato starch 18g, asparagine 2.6g (or peptone 3g), goes Skin Rhizoma Solani tuber osi 225g, 15, egg (removes 3 Ovum Gallus domesticus album)
2.2.2 compound method is as follows with reference to " code ": peeling potatoes 1) is wiped filamentation, adds potato starch, Tianmen Winter element (or peptone), skim milk are put water-bath in beaker and are boiled 40~60 minutes, and frequently stir, and make into pasty state.Treat Add the egg (eggshell is first cleaned and used 75% alcohol disinfecting) smashed when being cooled to 50 DEG C, after mixing, filter slagging-off with four layers of gauze. It is eventually adding glycerol and malachite green solution stirs, be sub-packed in the test tube of sterilizing.2) subpackage is had the test tube of culture medium Inclined-plane is put into, through flowing steam discontinuous sterilization 3 times, every day 1 time in putting flowing steam pot.65 DEG C of sterilizing 30min on the 1st, the 2nd, 3 Day, 75~80 DEG C of sterilizing 30min.
2.3 synthetic medium
2.3.1 basic components screening
Prepare by following composition: defatted milk powder 10g or 20g or 30g, yolk lecithin 5g or 10g or 15g, PPLO Agar powder 10g or 20g, asparagine 2.6g, peptone 2g, yeast powder 2g, glycerol 36ml, malachite green oxalate 0.6g, dehydrated alcohol 20ml, deionized water 1000ml.Then by defatted milk powder, yolk lecithin and PPLO agar powder, it is designed to table 1 by different proportion Shown 18 formula, are labeled as 1~18 (concrete ratio and numbering are shown in Table 1), set up Pei Shi culture medium to compare simultaneously.
2.3.2 preparation
In ratio shown in table 1 and amount, PPLO agar powder and malachite green oxalate 500ml are gone after water heating is fully dissolved, 116 DEG C of sterilizing 15min, are cooled to 60 DEG C as A liquid;By defatted milk powder, asparagine, peptone, yeast powder, glycerol, dimension life Element K 500ml deionized water, after 50 DEG C of heating in water bath dissolve, 0.22um filtration sterilization is as B liquid;By yolk lecithin with nothing The mode of bacterium operation fully dissolve with the dehydrated alcohol of 20ml in sterile vessel after as C liquid, then by A liquid and B liquid and C liquid After (V/V is 500:500:20) is sufficiently mixed, make culture medium slant with 8ml/ pipe standby.
2.4 seed liquor inoculations
By 2.1 seed liquor prepared, after making 10000 times of dilutions with normal saline, propping up by 0.2ml/, inoculation is made respectively Synthetic medium and Pei Shi medium slant, 37 DEG C cultivate 20 days.Observe each cultivation inclined-plane colony growth result.
The colony counting result (unit: CFU) of table 1 synthetic medium basic components screening
Note: seed liquor about 106CFU/ml。
Finding out from table 1 result, the ability of its 3 kinds of bacterium of cultivation of formula 14 is best, and apparently higher than comparison Pei Shi culture medium.
The screening of 2.5 culture medium somatomedin
Choosing the higher formula of breeding bacteria 14 as synthetic medium basic ingredient, wherein A liquid and C liquid are all by 2.3 Method is carried out.The glucose of various dose, sucrose, sodium thiosulfate, chlorine is added in ratio shown in table 2 and amount respectively in B liquid Change the somatomedin such as haemachrome, vitamin K.Then together use 500ml deionized water 50 together with basis B liquid composition in formula 14 After DEG C heating in water bath dissolves, 0.22um filtration sterilization uses as B liquid, then is made into solid medium by 2.3 methods.
The colony counting result (unit: CFU) of table 2 culture medium somatomedin screening
Note: access viable bacteria about 106The seed liquor of CFU/ml, Pei Shi comparison lot number is 151103.
Finding out from table 2 result, vitamin K is obvious to culture medium growth promoting function, and other somatomedin growth promoting function Inconspicuous.
2.6 somatomedin different amounts comparative tests
The somatomedin (vitamin K) that will filter out, by final concentration 0.005%, 0.01%, 0.02%, 0.05%, 0.1% adds in synthetic medium, then accesses seed liquor by 2.4 methods, cultivates 20 respectively for 37 DEG C, and observed result is shown in Table 3。
Table 3 different amounts somatomedin comparative test colony counting result (unit: CFU)
Note: access viable bacteria about 106The seed liquor of CFU/ml
Finding out from table 3 result, the vitamin K synthetic medium bacterium number when 0.01% is the highest.
2.7 synthetic mediums and Pei Shi culture medium comparative test
By formula 14 (adding the vitamin K of optimal dose), prepare 3 batches of synthetic mediums (lot number is respectively 201501, 201502 and 201503), together with 3 batches of Pei Shi culture medium (lot number is respectively 201511,201512 and 201513), A respectively it is sub-packed in Type test tube (8ml/ pipe).Prop up access dilution strain by 0.2ml/, cultivate 20 respectively for 37 DEG C.Respectively at cultivate 7 days, 10 days, 14 Day and 20 days, respectively observed result.
Table 4 culture medium comparative test result (unit: CFU)
Note: 3 kinds of bacterium strains: 1 be C68201,2 be C68202,3 for C68203, its seed liquor is each about 106CFU/ml; "/" represents that bacterium colony is invisible or naked eyes cannot count
Finding out from table 4 result, under same culture conditions, the breeding bacteria of 3 batches of synthetic mediums is all cultivated compared with 3 crowdes of Pei Shi The breeding bacteria of base is high, and incubation time shortens, additionally than the Pei Shi training with fresh skim milk with egg yolk preparation between batch Support base homogeneity preferable.
2.4 virulence tests cultivating thalline
Synthesis is cultivated lawn and the Pei Shi medium slant 37 DEG C cultivation lawn of 10 days that 37 DEG C of inclined-plane base is cultivated 7, After respectively scraping with aseptic soupspoon, weigh lawn weight, then with after appropriate normal saline dilution the mixing that suspends.Muscle note respectively Penetrate 8 week old SPF chicken 5,1ml/ (every 1ml viable bacteria Han 10mg), clinical observation 4 weeks, record survival condition;Intravenous injection respectively 8 week old SPF chickens 5,0.5ml/ (every 0.5ml viable bacteria Han 1mg), clinical observation 4 weeks, record survival condition.The results are shown in Table 5.
Thalline virulence determination result cultivated by table 5
Finding out from table 5 result, synthetic medium cultivates thalline and the Pei Shi culture medium culturing thalline of the 14 days poison of 7 days Power measurement result is basically identical, all meets the avain tuberculosis rhzomorph strain virulence standard that produces in " code ".
4 conclusions
4.1 with culture medium prescription 14 add 0.01% vitamin K composition synthetic medium breeding bacteria be above Pei Shi training Supporting base, and on synthetic medium, strain growth speed is better than Pei Shi culture medium, its incubation time is shorter than Pei Shi culture medium, and it is right SPF chicken toxicity test result is basically identical with Pei Shi culture medium, meets in " code " and " produces avain tuberculosis rhzomorph strain virulence mark Accurate " requirement.
Embodiment 2
The growth and breeding of other avain tuberculosis mycobacteria reference cultures is compared by synthetic medium with Pei Shi culture medium Result
The synthetic medium (201501 batches) of optional 1 batch of preparation and Pei Shi culture medium (201512 batches), purchased from country beast Other each 1 of avain tuberculosis mycobacteria reference culture freeze-drying lactobacillus of 22 strains that doctor's Culture Collection preserves, respectively After diluting with 1ml physiological saline solution, prop up inoculation above two medium slant with 0.2ml/ respectively, cultivate 14 for 37 DEG C, the phase Between 7,10 and 14 days observe observe growing state respectively.The results are shown in Table 6.
Table 6 different strain growing state
Note: "+" growth;"-" is without growth;
Finding out from table 6 result, synthetic medium is cultivated 10 at 37 DEG C, all can successfully turn out 22 strain strains, and antibacterial is raw Long speed is substantially better than Pei Shi culture medium.
Embodiment 3
Synthetic medium separates comparative result with Pei Shi culture medium to clinical pathological material of disease
Optional 1 batch of synthetic medium (201501 batches) and Pei Shi culture medium (201512 batches), to 5 parts of doubtful avain tuberculosis of clinic Sick pathological material of disease separates.Under Biohazard Safety Equipment environment, with sterile scissors sterile working picking pathological material of disease interior tissue about 0.1g, incite somebody to action It is put in the 1.5ml eppdorf pipe of the sterilizing containing rustless steel ball, is then respectively adding 200ul sterile saline, It is homogenized with tissue refiner (parameter is set as: 100r/min, and vibrate 1min, intermittently 1min, altogether 3min).Finally will tissue Homogenate respectively with after 200 μ l sterile salines dilutions, is transferred synthetic medium and Pei Shi culture medium with 200 μ l/ pipes respectively Each 1 pipe, cultivates 14~20 for 37 DEG C, observes slant culture result.The results are shown in Table 7.
Table 7 different culture media clinical isolates situation compares
Note: "+" colony growth and PCR be accredited as the positive;"-" is without growth;
Finding out from table 7 result, synthetic medium reaches 100% to clinical doubtful sample separation rate, and Pei Shi culture medium is only 80%.

Claims (2)

1., by a method for synthetic medium separation and Culture avain tuberculosis mycobacteria, it is characterized in that production fowl type tuberculin Avain tuberculosis mycobacteria reference culture C68201 strain, C68202 strain and the C68203 suitable normal saline dilution of strain freeze-drying lactobacillus After, inoculate with defatted milk powder, Ovum Gallus domesticus Flavus lecithin in the solid medium of synthetic medium primary raw material, cultivate 7 for 37 DEG C, make For preparation fowl type tuberculin first order seed.
2., according to the isolated culture method of a kind of avain tuberculosis mycobacteria described in claim 1, it is characterized in that used in it The formula (W/V) of synthetic medium is: defatted milk powder 20g, PPLO agar powder 20g, yolk lecithin 10g, asparagine 2.6g, Peptone 2g, yeast powder 2g, glycerol 36ml, vitamin K 0.1g, malachite green oxalate 0.6g, dehydrated alcohol 20ml, deionized water 1000ml.Its compound method is: PPLO agar powder and malachite green oxalate 500ml being gone after water heating is fully dissolved, 116 DEG C go out Bacterium 15min, is cooled to 60 DEG C as A liquid;Defatted milk powder, asparagine, peptone, yeast powder, glycerol, vitamin K are used 500ml deionized water, after 50 DEG C of heating in water bath dissolve, 0.22 μm filtration sterilization is as B liquid;By yolk lecithin with sterile working Mode fully dissolve with the dehydrated alcohol of 20ml in sterile vessel after as C liquid, then by A liquid and B liquid and C liquid, (V/V is After 500:500:20) being sufficiently mixed, make culture medium slant with 8ml/ pipe and use.
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CN113122484A (en) * 2021-06-03 2021-07-16 郑州安图生物工程股份有限公司 Freeze-drying protective agent for nontuberculous mycobacteria, and preparation method and preservation method thereof
CN113122484B (en) * 2021-06-03 2023-11-03 郑州安图生物工程股份有限公司 Freeze-drying protective agent for nontuberculous mycobacteria, and preparation method and preservation method thereof

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